PROJECT SUMMARY/ABSTRACT Ferritins are multi-subunit iron storage proteins that play a central role in minimizing iron toxicity and controlling intracellular iron homeostasis. In vertebrates, ferritins consist of hetero-polymeric complexes (isoferritins) of two different types of subunits, named L for Light, and H for Heavy. These two subunits co-assemble in various ratios with a tissue specific distribution, yielding a spectrum of isoferritins ranging from L-rich ferritin in the case of livers and spleens, to H-rich ferritin for hearts and brains. Surprisingly, despite the widespread occurrence of heteropolymer ferritins in tissues of vertebrates, very little is known about the complementary roles that H and L subunits play during iron uptake, iron mineralization and mobilization. After oxidation on the H-subunit catalytic centers, Fe(III) ions migrate to the protein cavity, where they aggregate at the nucleation sites of L-subunits and contribute to the growth of the iron mineral. The mechanism of iron mineralization, the influence of H- and L-subunits in iron core formation, and how certain amino acid side chains on these subunits influence iron mineral growth, remain largely unexplored. Once the inorganic iron core forms, the mechanism by which iron is mobilized from ferritin is also unclear and rather controversial. The generally accepted iron mobilization mechanism is believed to occur through ferritin proteolytic degradation. However, recent work from our lab suggest the existence of an auxiliary iron reductive mechanism that utilizes long-range electron transfer pathways, facilitated by the ferritin shell. The goals of this research proposal are to (1) elucidate the structure-function relationships and correlation between ferritin-subunit composition and iron core morphology, and to (2) investigate the physiological significance of the reductive dissolution of the ferritin iron core, under conditions close to physiological, and under controlled concentrations of oxygen. Specifically, we plan to (a) characterize the mineralization, structure, magnetism, and crystallinity of the iron core in isoferritins and in natural ferritin samples purified from animal organs, and (b) develop analytical methodologies to measure the in-vitro rates of iron reductive mobilization from ferritin under controlled concentrations of oxygen. To achieve this, a combination of molecular biology techniques, absorbance spectroscopy, isothermal titration calorimetry, Mssbauer spectroscopy, magnetometry, and a high-resolution (< 0.7 imaging capability) scanning and transmission electron microscopy will be employed. We believe that the results of this proposal should provide a detailed understanding of the roles that H- and L-subunits play in the morphology of the ferritin iron core, and help elucidate the role of the ferritin protein shell in controlling the iron mineral order, crystallinity, and access to iron chelators. It will also provide important insights into unresolved and fundamental questions related to the reductive mobilization of iron from ferritin in-vivo and its physiological significance.